Infectious diseases are the major cause of morbidity and mortality, accounting for a third of the deaths which occur in the world each year. In addition, infectious agents are directly responsible for at least 15% of new cancers, and they also seem to be involved in the pathophysiology of several chronic diseases (e.g. inflammatory, vascular and degenerative diseases). Traditional infectious diseases are also highly expensive in terms of health-associated costs of infected patients and loss in productivity at work.
The main strategies used to prevent infectious diseases are therapy and prophylaxis. Vaccination has become the most cost-effective measure to prevent infections. However, there are still many diseases for which vaccines are not yet available or the available vaccines are not completely satisfactory due to low efficacy, high reactogenicity, poor stability and/or high costs. Thus, there is still an urgent need for both new and improved vaccines.
Despite the fact that vaccines have traditionally been used for the prophylaxis of infectious diseases, recent findings suggest that they are also a powerful tool for the immunotherapy of transmissible diseases (e.g. viral hepatitis, Helicobacter pylori infections, herpes virus infections, etc.). In addition, vaccines can be used for the immune-therapy or immune-prophylaxis of autoimmune diseases, inflammatory diseases, tumours, allergies and for the control of fertility in human and/or animal populations. In particular, the last application seems to require the elicitation of efficient mucosal responses at the level of the reproductive tract.
Immunotherapy is the synonym for a therapy using the own immune system to attack the disorder or disease. For example immunotherapy for cancer stimulate the own immune system to attack the tumour. Typically, immunotherapy involves the use of cytokines or antibodies for treating cancer. All kind of cancer may be treated, like solid tumours or metastases. Immunotherapy is also possible for infectious diseases. Usually immunomodulators or adjuvants are required to induce a sufficient response and to stimulate the immune system of the treated individual.
Typically, costimulatory molecules are important to elicit a sufficient immune response and to obtain a satisfactory prophylactic or therapeutic treatment of a disease or disorder. For example, relevant costimulatory molecules are CD80 and CD86 as well as CD54 or CD40L. Further soluble molecules, in particular cytokine and chemokines play an important role. Thus, a balanced orchestra of costimulatory molecules is necessary to achieve favourable immune responses in the prophylaxis or treatment of various diseases and disorders, e.g. in vaccines.
Most infectious diseases are either restricted to the mucosal membranes or the etiologic agents need to transit the mucosa during the early steps of the infection. Therefore, it is desirable to obtain not only a systemic, but also a local mucosal immune response as a result of vaccination, thereby blocking both infection (i.e. colonization) and disease development. This may result in a more efficient protection against infection, facilitating also the eradication of diseases for which humans are the only reservoirs (i.e. blocking transmission to susceptible hosts). Parenterally-administered vaccines mainly stimulate systemic responses, whereas vaccines administered by a mucosal route mimic the immune response elicited by natural infections and can lead to efficient mucosal and systemic responses. Due to the apparent compartimentalization of the systemic and mucosal immune system, parenterally administered vaccines are less effective in protecting against mucosal pathogens (McGhee, J. R., Mestecky, J., Dertzbaugh, M. T., Eldridge, J. H., Hirasawa, M. and Kiyono, H. (1992) The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 10, 75-88). Thus, administration of immunogens through the mucosal route is required to achieve full protection. However, most of the available vaccines are administered through the parenteral route, thereby, eliciting a systemic immunity in the individual.
The administration of vaccines via the mucosal route offers several advantages over parenteral vaccination. These advantages include an ease of administration, the possibility of self-administration (e.g. by intranasal, rectal or oral application), the elimination of the chance of unwanted cross-infection due to the use of infected needles or non-sterile working, lower rates of side effects, higher acceptance by the public, better compliance of vaccination protocols (i.e. increment in the overall efficacy), simpler administration logistics and lower delivery costs, being particularly suitable for mass immunization programmes. However, the compartmentalisation at the level of the mucosal immune system has to be taken into consideration. In fact, immune responses which can be observed following intra-nasal vaccination may not necessarily occur after oral or intra-rectal immunisation. For example, oral vaccination may not stimulate efficient responses in the genitourinary and/or respiratory tracts.
Unfortunately, the delivery of antigens by the mucosal route is associated with a major problem, namely that antigens delivered by this route are generally poorly immunogenic. This is the result of different mechanisms, such as (i) accelerated antigen elimination by the non specific host clearance mechanisms (e.g. ciliar activity, peristaltism), (ii) antigen degradation by local enzymes, (iii) antigen alteration and/or structural modification as a result of extreme pH (e.g. acidic in the stomach, alkaline in the intestine), (iv) poor antigen penetration through the mucosa, (v) limited access of vaccine antigens to antigen presenting cells, and (vi) local peripheral tolerance.
To overcome these problems, different strategies have been used, such as antigen entrapment or association with physical or biological particles (e.g. microparticles, nanoparticles, bacterial ghosts), the use of virosomes or viral-like-particles, the use of liposomes or ISCOMS, the use of transgenic plants, antigen production by attenuated viral or bacterial carriers acting either as conventional vectors or as carriers for nucleic acid vaccines and/or their administration with mucosal adjuvants. However, despite the heavy body of experimental evidence generated in pre-clinical studies during the last years, almost no candidates have been transferred to the vaccine development pipeline.
The use of optimal adjuvants plays a crucial role in vaccination. Antigens administered without adjuvant only rarely mediate an adequate immune response. In addition, not only the strength but also the quality of the elicited immune response matters. Stimulation of an incorrect immunization pattern may lead to immunopathological reactions and exacerbation of the symptoms of infection. In this context, the adjuvant can help to assist the desired immune response. In other words, an adjuvant can modulate the immune response or redirect the immune response to balance the immune response in the desired direction.
One major obstacle is to be able to induce systemic as well as mucosal responses to mucosal vaccines. Furthermore, other approaches, such as mucosally induced tolerance also seems to be a promising form of immunomodulation for treating certain autoimmune diseases and allergies. Improved biochemical techniques have allowed purifying and/or constructing of new and well characterised adjuvants. Recent advances in our understanding of the immune system, most particularly with respect to early pro-inflammatory signals, have led to the identification of new biological targets for vaccine adjuvants. In particular, one can now choose immuno-potentiating adjuvants for Protein- and DNA-based vaccines, able to selectively induce T helper (Th)-1 and/or Th2 responses, according to the vaccine target and the desired immune response. As our knowledge of the cell types and cytokines interacting in the immune responses increases, so does our understanding of the mode of action of adjuvants, as well as the way in which they produce adverse effects.
Substances referred to as “adjuvants” are those which are added and/or co-formulated in an immunization to the actual antigen (i.e. the substance which provokes the desired immune response) in order to enhance the humoral and/or cell-mediated immune response (“Lexikon der Biochemie und Molekularbiologie”, 1. Band, Spektrum, Akademischer Verlag 1995). That is, adjuvants are compounds having immunopotentiating properties, in particular, when co-administered with antigens. The use of many adjuvants is based solely on experience, and the effect can neither be accurately explained nor predicted. The following groups of adjuvants are traditionally used in particular: aluminum hydroxide, emulsions of mineral oils, saponins, detergents, silicon compounds, thiourea, endotoxins of gram-negative bacteria, exotoxins of gram-positive bacteria, killed or attenuated living bacteria or parts thereof.
As noted above, administration of antigen in combination with suitable adjuvants generally permits use of a much smaller amount of antigen and increases the antibody titer. However, most of the adjuvants presently used in vaccination suffer in their properties regarding (bio)safety, biodegradability, stability, ease of mixing and use, broad range of antigens and administration routes as well as on economic and reliable production.
An overview over the presently known mucosal adjuvants and delivery systems, e.g. the above mentioned particles, ICOMS, liposomes and viral-like particles, for protein-DNA- and RNA-based vaccines is given in Vajdy et al., Immunol. Cell Biol., 2004, 82, 617-627. Therein the currently available approaches in immunopentiation of mucosal vaccines are discussed.
That is, various mucosal adjuvants have been described which should serve as an alternative for the adjuvants useful for systemic administration, e.g. see Vajdy et al., supra. These mucosal adjuvants include heat labile enterotoxin and detoxified mutants thereof. In particular, genetically detoxified mutants of heat labile enterotoxin of E. coli have been developed as useful mucosal adjuvants. Moreover, cholera toxin of vibrio cholera is known as an adjuvant useful for mucosal vaccination. Further, the application of unmethylated CpG dinucleotides has been described. It was shown that CpG can bias the immune response towards a Th1 response and can modulate pre-existing immune responses. Saponins are also described as immunomodulatory substances, predominantly via the induction of specific cytokines which then modulate and/or activate the immune response.
In addition, as adjuvants which may be useful in mucosal vaccination the following have been described:
The MALP-2 molecule and Bisaxcyloxypropylcysteine-conjugates thereof, e.g. a Bispalmitoyloxypropylcysteine-PEG molecule is known to represent potent stimulants for macrophages. The usefulness of MALP-2 as an adjuvant was shown previously, see e.g. WO2004/009125 and WO2003/084568. In particular, it was demonstrated that MALP-2 can act as an effective mucosal adjuvant enhancing the mucosal immune response, e.g. fostering an enhanced expression of antigen-specific IgA antibodies.
Furthermore, it was shown that MALP-2 can activate dendritic cells and B-cells, both play an important rule in the induction of a specific humoral immune response. In addition preliminary studies demonstrate that a combination for biologically active HIV-1 that protein and synthetic MALP-2 may be a promising vaccine with the MALP-2 component as an effective mucosal adjuvant.
Unfortunately, most of the compounds described above being useful as mucosal adjuvants are not utilisable due to their intrinsic toxicity, e.g. retrograde homing to neuronal tissues of bacterial toxoids and/or toxins at/in the derivatives after nasal vaccination. Other reasons for the inapplicability of most of the known adjuvants are their lack in (bio)safety, biodegradability, stability, broad range of application etc as noted above.
Thus, none of these previously described mucosal adjuvants have been approved yet, but, today, only two systemic adjuvants received approval to be administered to humans and, hence, are used for the preparation of human vaccines. These adjuvants are Alum and MF59. However, both are not effective as mucosal adjuvants.
There has been an intensive search in recent years for novel adjuvants, including those for the mucosal administration route. Only a few substances have been found to be able to enhance mucosal responses. Among these, some act as carriers to which the antigens must be bound or fused thereto. Far fewer universally employable “true” adjuvants which are admixed to the antigens have been found, as outlined above.
Glycopolymers are sugar-containing compounds, which may consist of repeating units of mono- or oligosaccharides (chemical glycopolymers) or which are sugar-containing macromolecules which in aqueous suspensions form polymeric aggregates (physical glycopolymers). Of course, the chemical glycopolymers may also be able to form physical glycopolymers. The glycoconjugates, glycolipids and glycoproteins, belong to the latter class of physical glycopolymers, as the most important group. The biological importance of this class has become evident only in the last 20 years. Glycolipids are essential constituents of cellular membranes with a large of number of functions. They may act as receptors, may provide specific contact, be important for cell aggregation and dissociation, and may transmit and receive signals, for example, to initiate cell division. Cerebrosides and monoacylglycosides belong to the simple glycolipids, and gangliosides belong to the more complex glycolipids, which usually have specialized functions as e.g. as cell surface receptors. Many glycolipids are known to modulate the immune response. In common nomenclature also bacterial lipopolysaccharides (LPS, endotoxins) belong to this class. LPS induce a variety of biological effects in mammals like activation of mononuclear cells to produce cytokines such as tumor necrosis factor and interleukins. Glycoproteins, i.e., proteins with a covalently linked carbohydrate moiety, are also ubiquitous in nature. They are found in cell membranes and inside cells, in the cytoplasm as well as in subcellular organelles and in extracellular fluids [Brandenburg K., et al., Carbohydrate Research 2003, 338 (23), 2477-2489].
Glyco glycero lipids are known to be an important class of glycolipids. These lipids can be found as important constituents in cell membranes. In principle, these lipids consist of a carbohydrate head-group as polar head while the unpolar tail is build up by glycerol esters or ethers of alkyl or acyl chains [Sing, N., et al., J. Immunol 1999, 163 (5), 2373-2377]. Lipids with small headgroups (1-3 carbohydrate units in the head) can be found as major components of the cell membrane, whereas the more complex lipids are involved in cell surface recognition processes [Brandenburg, supra].
In view of the known adjuvants described above, there is still a need to provide new compounds useful as adjuvants, particularly as mucosal adjuvants, and/or as vaccines. In particular, there is a need for mucosal adjuvants which can elicit a strong immune response which represent a balanced and/or adjusted immune response involving both humoral and cellular components, thus, allowing effective prophylaxis or treatment of various diseases and conditions, specifically of infectious diseases or cancer.
Thus, the object of the present invention is the provision of mucosal adjuvants which can elicit and/or enhance and/or modulate (pre-existing) immune response in an individual or subject. In particular, the invention was based on the object of developing a range of novel, highly active adjuvants, particularly mucosal adjuvants which are non-toxic for humans and which can be employed with a wide variety of active ingredients to be assisted in conventional or novel vaccines such as, in particular, prophylactic or therapeutic vaccines, including cancer and DNA vaccines.
Citation of any document herein is not intended as admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application.